This study describes the transcriptional response of P. aeruginosa PAO1 to low-Earth orbit environmental conditions. Our aim was to assess whether the microgravity environment of spaceflight could induce virulence traits in P. aeruginosa. To this end, P. aeruginosa cultures were grown in space, and the expression profile was compared with ground control samples (both in biological triplicate). Characterization of bacterial behavior in the microgravity environment of spaceflight is of importance towards risk assessment and prevention of infectious disease during long-term missions. Further, this research field unveils new insights into connections between low fluid-shear regions encountered by pathogens during their natural infection process in vivo, and bacterial virulence. This study is the first to characterize the global transcriptomic and proteomic response of an opportunistic pathogen that is actually found in the space habitat, Pseudomonas aeruginosa. Overall, P. aeruginosa responded to spaceflight conditions through differential regulation of 167 genes and 28 proteins, with Hfq identified as a global transcriptional regulator in the response to this environment. Since Hfq was also induced in spaceflight-grown Salmonella typhimurium, Hfq represents the first spaceflight-induced regulator across the bacterial species border. The major P. aeruginosa virulence-related genes induced in spaceflight conditions were the lecA and lecB lectins and the rhamnosyltransferase (rhlA), involved in the production of rhamnolipids. The transcriptional response of spaceflight-grown P. aeruginosa was compared with our previous data of this organism grown in microgravity-analogue conditions using the rotating wall vessel (RWV) bioreactor technology. Interesting similarities were observed, among others with regard to Hfq regulation and oxygen utilization. While LSMMG-grown P. aeruginosa mainly induced genes involved in microaerophilic metabolism, P. aeruginosa cultured in spaceflight adopted an anaerobic mode of growth, in which denitrification was presumably most prominent. Differences in hardware between spaceflight and LSMMG experiments, in combination with more pronounced low fluid shear and mixing in spaceflight when compared to LSMMG conditions, were hypothesized to be at the origin of these observations. Collectively, our data suggest that spaceflight conditions could induce the transition of P. aeruginosa from an opportunistic organism to potential pathogen, results that are of importance for infectious disease risk assessment and prevention, both during spaceflight missions and in the clinic.

Anticipating the risk for infectious disease during space exploration and habitation is a critical factor to ensure safety health and performance of the crewmembers. As a ubiquitous environmental organism that is occasionally part of the human flora Pseudomonas aeruginosa could pose a health hazard for the immuno-compromised astronauts. In order to gain insights in the behavior of P. aeruginosa in spaceflight conditions two spaceflight-analogue culture systems i.e. the rotating wall vessel (RWV) and the random position machine (RPM) were used. Microarray analysis of P. aeruginosa PAO1 grown in the low shear modeled microgravity (LSMMG) environment of the RWV compared to the normal gravity control (NG) revealed a regulatory role for AlgU (RpoE). Specifically P. aeruginosa cultured in LSMMG exhibited increased alginate production and up-regulation of AlgU-controlled transcripts including those encoding stress-related proteins. This study also shows the involvement of Hfq in the LSMMG response consistent with its previously identified role in the Salmonella LSMMG- and spaceflight response. Furthermore cultivation in LSMMG increased heat and oxidative stress resistance and caused a decrease in the culture oxygen transfer rate. Interestingly the global transcriptional response of P. aeruginosa grown in the RPM was similar to that in NG. The possible role of differences in fluid mixing between the RWV and RPM is discussed with the overall collective data favoring the RWV as the optimal model to study the LSMMG-response of suspended cells. This study represents a first step towards the identification of specific virulence mechanisms of P. aeruginosa activated in response to spaceflight-analogue conditions and could direct future research regarding the risk assessment and prevention of Pseudomonas infections for the crew in flight and the general public.

Genome-wide transcriptional profiling showed that reducing gravity levels in the International Space Station (ISS) causes important alterations in Drosophila gene expression intimately linked to imposed spaceflight-related environmental constrains during Drosophila metamorphosis. However simulation experiments on ground testing space-related environmental constraints show differential responses. Curiously although particular genes are not common in the different experiments the same GO groups including a large multigene family related with behavior stress response and organogenesis are over represented in them. A global and integrative analysis using the gene expression dynamics inspector (GEDI) self-organizing maps reveals different degrees in the responses of the transcriptome when using different environmental conditions or microgravity/hypergravity simulation devices. These results suggest that the transcriptome is finely tuned to normal gravity. In regular environmental conditions the alteration of this constant parameter on Earth can have mild effects on gene expression but when environmental conditions are far from optimal the gene expression is much more intense and consistent effects.

Changes in the physical environment modulate cell responses and may lead to the impairment or even failure of tissue function as a result of mechanotransduction processes. It has been suggested that this situation occurs in some age-related diseases and some pathological conditions observed in space such as cardiovascular deconditioning bone loss muscle atrophy and impaired immune responses. All of these are associated with endothelial dysfunction but the precise mechanism is still unclear. We used the microarray approach to obtain insights into the mechanism responsible for endothelial dysfunction by taking advantage of the challenging environment of gravitational unloading onboard the International Space Station. The effects of gravitational unloading on HUVEC gene expression were investigated by means of cDNA microarray analyses of six randomly chosen samples (three for each of the two conditions of spaceflight and 1g) using Affymetrix Gene Human 1.0 ST Arrays

Larvae-Pupae transition flies (Drosophila) were recovered and transport for 3 days at 12-14C to arrest development until the launch site then exposed to RT (18-20C) for some hours including the launch and trip to the International Space Station then pupae were exposed to microgravity in the ISS for 4 days and a half at 22C. Finally pupae were fixed on acetone and frozen until recovery on Earth. Four groups of samples: 1 ISS (+ground control) as described 2 RPM (microgravity simulator on Earth) as described 3 RPM without constrains (No MAMBA container and only 5 days exposure without cold transport) and 4 centrifuge 10g without constrains control.

Arabidopsis thaliana was evaluated for its response to the spaceflight environment in three replicated experiments on the International Space Station. Two approaches were used; GFP reporter genes were used to collect gene expression data in real time within unique GFP imaging hardware and plants were harvested on orbit to RNAlater for subsequent analyses of gene expression with using Affymetrix and SAGE transcriptome analyses. Three tissue types were examined (leaves hypocotyls and roots) and compared to analyses conducted with whole plants. Transcriptome analyses with whole plants suggested that the spaceflight environment had little impact on the transcriptome of arabidopsis however closer examination of selected tissues revealed that there are a number of tissue-specific responses that arabidopsis employs to respond to this novel environment

BACKGROUND: Ionizing radiation (IR) can be extremely harmful for human cells since an improper DNA-damage response (DDR) to IR can contribute to carcinogenesis initiation. Perturbations in DDR pathway can originate from alteration in the functionality of the microRNA-mediated gene regulation being microRNAs (miRNAs) small noncoding RNA that act as post-transcriptional regulators of gene expression. In this study we gained insight into the role of miRNAs in the regulation of DDR to IR under microgravity a condition of weightlessness experienced by astronauts during space missions which could have a synergistic action on cells increasing the risk of radiation exposure. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed miRNA expression profile of human peripheral blood lymphocytes (PBL) incubated for 4 and 24 h in normal gravity (1 g) and in modeled microgravity (MMG) during the repair time after irradiation with 0.2 and 2Gy of gamma-rays. Our results show that MMG alters miRNA expression signature of irradiated PBL by decreasing the number of radio-responsive miRNAs. Moreover let-7i* miR-7 miR-7-1* miR-27a miR-144 miR-200a miR-598 miR-650 are deregulated by the combined action of radiation and MMG. Integrated analyses of miRNA and mRNA expression profiles carried out on PBL of the same donors identified significant miRNA-mRNA anti-correlations of DDR pathway. Gene Ontology analysis reports that the biological category of Response to DNA damage is enriched when PBL are incubated in 1 g but not in MMG. Moreover some anti-correlated genes of p53-pathway show a different expression level between 1 g and MMG. Functional validation assays using luciferase reporter constructs confirmed miRNA-mRNA interactions derived from target prediction analyses. CONCLUSIONS/SIGNIFICANCE: On the whole by integrating the transcriptome and microRNome we provide evidence that modeled microgravity can affects the DNA-damage response to IR in human PBL. Overall Design: Gene expression signature was defined in PBL irradiated with gamma-rays (2.0 Gy) and incubated in modeled microgravity (mmg) and in parallel ground conditions (1g) for 24h. Five independent experiments were performed for each donor to address which mRNAs were regulated on IR stress. The level of each transcript was represented as Log2.

BACKGROUND: Ionizing radiation (IR) can be extremely harmful for human cells since an improper DNA-damage response (DDR) to IR can contribute to carcinogenesis initiation. Perturbations in DDR pathway can originate from alteration in the functionality of the microRNA-mediated gene regulation being microRNAs (miRNAs) small noncoding RNA that act as post-transcriptional regulators of gene expression. In this study we gained insight into the role of miRNAs in the regulation of DDR to IR under microgravity a condition of weightlessness experienced by astronauts during space missions which could have a synergistic action on cells increasing the risk of radiation exposure. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed miRNA expression profile of human peripheral blood lymphocytes (PBL) incubated for 4 and 24 h in normal gravity (1 g) and in modeled microgravity (MMG) during the repair time after irradiation with 0.2 and 2Gy of gamma-rays. Our results show that MMG alters miRNA expression signature of irradiated PBL by decreasing the number of radio-responsive miRNAs. Moreover let-7i* miR-7 miR-7-1* miR-27a miR-144 miR-200a miR-598 miR-650 are deregulated by the combined action of radiation and MMG. Integrated analyses of miRNA and mRNA expression profiles carried out on PBL of the same donors identified significant miRNA-mRNA anti-correlations of DDR pathway. Gene Ontology analysis reports that the biological category of Response to DNA damage is enriched when PBL are incubated in 1 g but not in MMG. Moreover some anti-correlated genes of p53-pathway show a different expression level between 1 g and MMG. Functional validation assays using luciferase reporter constructs confirmed miRNA-mRNA interactions derived from target prediction analyses. CONCLUSIONS/SIGNIFICANCE: On the whole by integrating the transcriptome and microRNome we provide evidence that modeled microgravity can affects the DNA-damage response to IR in human PBL. Overall Design: Gene expression signature was defined in PBL irradiated with gamma-rays (2.0 Gy) and incubated in modeled microgravity (mmg) and in parallel ground conditions (1g) for 24h. Five independent experiments were performed for each donor to address which mRNAs were regulated on IR stress. The level of each transcript was represented as Log2.

Genome-wide transcriptional profiling showed that reducing gravity levels in the International Space Station (ISS) causes important alterations in Drosophila gene expression intimately linked to imposed spaceflight-related environmental constrains during Drosophila metamorphosis. However simulation experiments on ground testing space-related environmental constraints show differential responses. Curiously although particular genes are not common in the different experiments the same GO groups including a large multigene family related with behavior stress response and organogenesis are over represented in them. A global and integrative analysis using the gene expression dynamics inspector (GEDI) self-organizing maps reveals different degrees in the responses of the transcriptome when using different environmental conditions or microgravity/hypergravity simulation devices. These results suggest that the transcriptome is finely tuned to normal gravity. In regular environmental conditions the alteration of this constant parameter on Earth can have mild effects on gene expression but when environmental conditions are far from optimal the gene expression is much more intense and consistent effects.

The effect of microgravity on gene expression in C.elegans was comprehensively analysed by DNA microarray. This is the first DNA microarray analysis for C.elegans grown under microgravity. Hyper gravity and clinorotation experiments were performed as reference against the flight experiment.